WO2020248007A1 - Prostate cancer detection - Google Patents

Abstract

A method of selective capturing target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid is disclosed. The method comprises providing a sample of urine or a urine derived fluid; providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; and contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell-derived exosomes from the urine (if present) to the cell capture surface.

Description

PROSTATE CANCER DETECTION

PRIORITY DOCUMENT

[0001] The present application claims priority from Australian Provisional Patent Application

No. 2019902054 titled“PROSTATE CANCER DETECTION” and fded on 12 June 2019, the content of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to methods and devices for selectively capturing prostate cancer cells from urine samples.

BACKGROUND

[0003] Prostate cancer is the second most common cancer in men and a leading cause for male cancer death. The current blood test for detecting prostate cancers measures a molecule called prostate specific antigen (PSA) but this test unfortunately has many limitations including a very high rate of false positives and false negatives. Furthermore, current tests do not distinguish between patients with aggressive cancers that are likely to spread, cause death and require treatment and prostate cancers which are indolent, do not cause symptomatic disease or affect lifespan and do not require treatment.

[0004] Australia had the highest incidence of prostate cancer in 2012 ahead of USA and Canada. Over one million men go through prostate biopsies per annum in the United States. Although prostate biopsy is currently the gold standard for diagnosis of prostate cancers, it is painful and invasive with serious side effects. More than half of the biopsies turn out to be negative for prostate cancer for a variety of different reasons. For example, elevated prostate specific antigen (PSA) levels are often caused by conditions other than cancer and this is one reason for the high rate of false positives. Also, biopsy needles can miss the prostate tumor foci and this results in false negatives (Afshar-Oromieh et al., 2013; Fujita el al., 2009a; Nickens et al., 2015a; Pal et al., 2016a). Therefore, it is of great importance, for health systems and the wellbeing of the aging population, to develop a screening technique with a better negative and positive predictive value for prostate cancer diagnosis and which can predict the aggressiveness of the cancer and the need for treatment.

[0005] Previously, researchers have tried to produce non-invasive methods for prostate cancer diagnosis using patient urine samples. It has been recognised that prostate cancer cells could be shed in urine after digital rectal examination or prostatic massage. Different urine based molecular assays such as Prostate cancer antigen 3 (PCA3), Transmembrane protease, serine 2 enzyme (TMPRSS2-ERG) (D. Hessels & J. A. Schalken, 2013) and blood based tests like Prostate Health Index [PHI], four kallikreins score [4K] and CTC assays have been used for detecting prostate cancer with disputed benefits (Loeb & Catalona, 2014; Sartori & Chan, 2014; Voigt, Zappala, Vaughan, & Wein, 2014). Moreover, Urine Prostate Cancer Marker Panel (UCMP) has been used after digital rectal examination for detection of cells at the single cell level. However, this approach required filtration of urine samples and a large number of steps to process the samples (Nickens et al., 2015a). Overall, these approaches return unsatisfactory sensitivity with respect to prostate cancer cells, even with post-digital rectal examination urine and require a great deal of preparation.

[0006] There is a need for a non-invasive, cost effective and/or user-friendly platform to diagnose prostate cancer and/or provide prognostic information.

[0007] In particular, aggressive prostate cancer cells are more likely to grow, spread and metastasise and hence more likely to appear in urine. Building on this distinctive behaviour of aggressive prostate cancer, the capture of prostate cancer cells naturally shed in urine is likely to provide prognostic information.

SUMMARY

[0008] The present disclosure is based on the inventors' development of a new diagnostic method and device that combines prostate specific immunocapture and cancer specific photosensitisers to capture whole prostate cancer cells from voided urine samples in order to provide a diagnostic method capable of detecting or ruling out prostate cancer with a specificity that is better than the current gold standard PSA blood test.

[0009] In a first aspect, the present disclosure provides a method of selectively capturing target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid, the method comprising:

providing a sample of urine or a urine derived fluid;

providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; and

contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell- derived exosomes from the urine (if present) to the cell capture surface.

[0010] In embodiments of the first aspect, the method further comprises detecting target prostate cancer cells or target prostate cancer cell-derived exosomes on the one or more cell capture surface. [001 1] In certain embodiments, captured prostate cancer cells can be detected using a cancer specific photosensitiser composition. Cancer specific photosensitiser compositions that can be used to detect captured prostate cancer cells include, but are not limited to, compositions comprising 5 -aminolevulinic acid (ALA 5), hexaminolevulinate (HAL) and/or hypericin. In certain of these embodiments, the captured prostate cancer cells can additionally be detected using a luminescent cell nucleus stain composition.

[0012] Alternatively, or in addition, in certain embodiments captured prostate cancer cells or prostate cancer cell-derived exosomes can be detected by using other techniques known in the art including, but not limited to, antibody staining and genetic analysis. For example, the genetic content of the captured prostate cancer cells or prostate cancer cell-derived exosomes can be tested using PCR to detect specific micro RNAs and DNA fragments of prognostic value. By way of example, following the method of Tinzl, an amplification assay (uPM3™) can be used to detect DD3^PCA3 RNA in captured prostate cancer cells or exosomes (Tinzl et al., 2004).

[0013] It will be apparent from the foregoing discussion that the method of the first aspect can be applied to the diagnosis, prognosis and/or monitoring of prostate cancer in a patient. Thus, in a second aspect the present disclosure provides a method for diagnosing, prognosing or monitoring prostate cancer in a mammal, the method comprising:

providing a sample of urine or a urine derived fluid obtained from the mammal;

providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome;

contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell- derived exosomes from the urine (if present) to the cell capture surface; and

detecting any target prostate cancer cells or target prostate cancer cell-derived exosomes bound to the cell capture surface.

[0014] In embodiments of the second aspect, the step of detecting the target prostate cancer cells or target prostate cancer cell-derived exosomes bound to the cell capture surface comprises detecting the target prostate cancer cells using a cancer specific photosensitiser composition. Cancer specific photosensitiser compositions that can be used to detect captured prostate cancer cells include, but are not limited to, compositions comprising 5-aminolevulinic acid (ALA 5), hexaminolevulinate (HAL) and/or hypericin. In certain of these embodiments, the captured prostate cancer cells can additionally be detected using a luminescent cell nucleus stain composition.

[0015] Alternatively, or in addition, in certain embodiments, captured prostate cancer cells or target prostate cancer cell-derived exosomes can be detected by using other techniques known in the art including, but not limited to, antibody staining and genetic analysis. For example, the genetic content of the captured prostate cancer cells or target prostate cancer cell-derived exosomes can be tested using PCR to detect specific micro RNAs and DNA fragments of prognostic value.

[0016] It will also be apparent from the foregoing discussion that the method of the first aspect can be used to capture or immobilise prostate cancer cells or target prostate cancer cell-derived exosomes on a surface. Thus, in a third aspect the present disclosure provides a method of immobilising target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid on a substrate surface, the method comprising:

providing a sample of urine or a urine derived fluid;

providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; and

contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell- derived exosomes from the urine (if present) to the cell capture surface.

[0017] In a fourth aspect, also provided herein is a device for selective capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid, the device comprising a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome.

[0018] In a fifth aspect, provided herein is a microfluidic device for selective capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid, the device comprising a substrate having one or more cell capture micro-channel, each cell capture micro-channel comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome.

[0019] In certain embodiments of the first to fifth aspects, the cell capture surface comprises a functionalized film on the substrate and the one or more target prostate cancer cell selective binding agent(s) is/ are covalently bound to the functionalized film.

[0020] In certain embodiments of the first to fifth aspects, the target prostate cancer cell selective binding agents comprise one or more PSMA antibody or functional equivalent thereof. BRIEF DESCRIPTION OF DRAWINGS

[0021] Embodiments of the present disclosure will be discussed with reference to the accompanying figures wherein:

[0022] Figure 1 demonstrates cell capture on PiPOx functionalized surfaces;

[0023] Figure 2 shows a schematic showing functionalizing plasma polymerised oxazoline (POX) coated microchannels with block proteins and prostate specific membrane antigen (PSMA) for selective capture of prostate cancer cells;

[0024] Figure 3 shows plots showing the effect of hexaminolevulinate (F1AL) concentration, incubation time and temperature on the fluorescence of prostate cancer LNCaP cells and non-cancer prostate PNT2 cells after A) 0.5h, B) 1h and C) 2h;

[0025] Figure 4 shows the effect of NuclearRed on cell fluorescence intensity that changes after 1 hour incubation with various HAL concentrations in PBS with or without NuclearRed staining. Cells were measured under a fluorescence microscope. A) Mean fluorescence intensity B) Percentages of difference; C) Fluorescence microscopy images of prostate cancer LNCaP cells and non-cancer PNT2 cells.

Magnification 5x;

[0026] Figure 5 shows a. Western blot analysis on prostate PNT2 and LnCAP cells using PSMA and EpCAM antibodies; b. Schematic of PSMA antibody which presents cells are targeting the extracellular domain; and c. Demonstrates the capture rate of LNCaP and PNT2 cell lines with various PSMA antibody (107-1A4) concentrations, which is higher for 10mg/ml compared to the higher ones;

[0027] Figure 6 shows a plot demonstrating the capture rate of LNCaP and PNT2 cell lines with PSMA antibody (107-1A4) at 10mg/ml in PBS and urine media, where LNCaP capture rate is about 90% in both media;

[0028] Figure 7 I. shows how PNT2 and LNCaP cells were stained where‘A’ = PNT2 without stain,

‘B’ = PNT2 + HAL (50 mmol/L) and‘C’= [(LNCaP+ HAL (50 mihoI/L) + Nuclear-Red (OA- mmol.L-¹)] + [PNT2 + HAL (50 mmol/L)]; II. Demonstrates the capture rate of LNCaP and PNT2 cell lines with 10mg/ml of PSMA antibody (107-1A4); and III. Presents absolute cell numbers in each channel for A, B and C;

[0029] Figure 8 demonstrates the comparison in capture rate of LNCaP and PNT2 cell lines with 107- 1A4, ab66912 and EpCAM (a bladder specific antibody); and [0030] Figure 9 shows histogram data obtained from the PpIX fluorescence images of different ratios of cancer and non-cancer cells mixed. Cells were incubated with 50mM HAL in 37°C for 1h and 2h respectively.

DESCRIPTION OF EMBODIMENTS

[0031] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. "Consisting essentially of' shall mean excluding other elements of any essential significance to the combination. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0032] The term "about" or "approximately" means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 fold, and more preferably within 2 fold, of a value. Unless otherwise stated, the term 'about' means within an acceptable error range for the particular value, such as ± 1-20%, preferably ± 1-10% and more preferably ±1-5%.

[0033] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0034] Disclosed herein is a method for selectively capturing target prostate cancer cells capable of binding one or more target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid. The method comprises providing a sample of urine or a urine derived fluid, providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome, and contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell-derived exosomes from the urine (if present) to the cell capture surface. [0035] The method may further comprise detecting target prostate cancer cells or target prostate cancer cell-derived exosomes on the one or more cell capture surface, as described in more detail later.

[0036] Advantageously, the substrate that is used to capture target prostate cancer cells or target prostate cancer cell-derived exosomes can be part of a point of care device capable of selective prostate cancer cell or exosome capture from urine. Thus, also provided herein is a device for selective capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid. The device comprises a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agents capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome. This then provides a rapid and reliable method and device for screening patients for prostate cancer and/or monitoring patients for prostate cancer.

[0037] As used herein, the term "prostate cancer", and similar terms, means a malignant tumor of the prostate gland. Almost all prostate cancers are adenocarcinomas, although other types of prostate cancer include sarcomas, small cell carcinomas, neuroendocrine tumors (other than small cell carcinomas), and transitional cell carcinomas. The methods and devices disclosed herein can be configured to capture any one or more of these prostate cancer cells from urine. The methods and devices disclosed herein can also be configured to capture any one or more prostate cancer cell-derived exosome from urine. Exosomes are small extracellular vesicles (EV) ranging from 50 to 150 nm in diameter. Exosomes have a double membrane structure with various cargo contents, such as miRNAs, mRNAs, proteins, lipids and viral particles. Exosomes are released by the exocytosis of multivesicular bodies (MVBs). Exosomes are present in human body fluids such as the blood, urine and saliva. Exosomes from cancer stem cells support prostate cancer tumorigenesis through promoting angiogenesis. Exosomes from tumor microenvironments are important regulators to enhance prostate cell survival, proliferation, angiogenesis and the evasion of immune surveillance, which contribute to prostate cancer progression. The potential of exosomes to provide candidate biomarkers for prostate cancer has been studied (Soekmadji et al.).

[0038] In certain embodiments, the cell capture surface comprises a functionalized film on the substrate and the one or more prostate cancer cell or exosome selective binding agent(s) is/are covalently bound to the functionalized film. In these embodiments, any suitable substrate can be used provided a

functionalized film can be formed on the surface thereof and can be retained on the surface thereof under typical operating conditions. Suitable substrate materials include glass, silicon, ceramics, metals, plastics, polymeric materials, paper, paper laminates, cellulose, carbon fibre, biomaterials, surfaces comprising biological molecules, surfaces comprising small organic molecules, surfaces comprising inorganic molecules, etc. The plastic may be selected from the group consisting of: polycarbonate, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyethylene terephthalate; polyethylene naphthalene dicarboxylate, tetrafluoroethylene- hexafluoropropylene copolymers, polyvinyl-difluoride, nylon, polyvinylchloride, copolymers of the aforementioned, and mixtures of the aforementioned. In some embodiments, the substrate is glass. In other embodiments, the substrate is silicon.

[0039] The substrate has one or more cell capture surface. The one or more cell capture surface can be formed in one or more feature on a surface of the substrate. The one or more feature may be in the form of a well, such as in a 96 well plate, or they may be one or more fluid flow path of any size, geometry or configuration. The one or more fluid flow path may be in the form of one or more channel (open or enclosed) such as channels commonly used in "flow through" type diagnostic devices. In certain embodiments, the substrate contains microfluidic features, such as microfluidic channels in a microfluidic device. As used herein, the term "micro fluidic", and variants thereof, means that the chip, device, apparatus, substrate or related apparatus containing fluid control features that have at least one dimension that is sub-millimetre and, typically less than 100 mm, and greater than 1 mm. Furthermore, the term "microchannel", and variants thereof, means a channel having at least one dimension that is sub- millimetre and, typically less than 100 mm, and greater than 1 mm.

[0040] In certain embodiments, the device is a microfluidic device. Thus, provided herein is a microfluidic device for selective capture of target prostate cancer cells or target prostate cancer cell- derived exosomes from urine or a urine derived fluid, the device comprising a substrate having one or more cell capture micro-channel, each cell capture micro-channel comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome.

[0041] In certain embodiments, the micro-channel is as described in U.S. Patent Application

Publication No. 2011/0294187, which is incorporated herein by reference in its entirety. Specifically, the micro-channel can be defined with three dimensional (3D) patterns. This 3D patterning allows one to affect the flow profile within the micro-channel, which in turn enhances the interaction between the flowing sample urine solution and the cell capture surface, and subsequently significantly increases the cell capture efficiency. In some embodiments, the micro-channel surface is made from

polymethylmethacrylate (PMMA).

[0042] The methods and devices described herein are used for the selective capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid. Urine is a complex mixture containing water, salts, urea, debris, proteins, cells, and this complexity typically presents a challenge for urine cytologists. In contrast, the selectivity of the methods and devices described herein means that cancer cells or target prostate cancer cell-derived exosomes are selectively

immobilised, thus overcoming problems of complexity, extracellularity and cytomorphology. [0043] As discussed in more detail later, the selectivity of the methods and devices described herein is brought about by using one or more binding agents that selectively bind cancer cells or prostate cancer cell-derived exosomes of interest and, more specifically, selectively bind biological markers of the cancer cells or prostate cancer cell-derived exosomes of interest. As used herein, the term "selective capture" and similar terms when used in relation to the capture of target prostate cancer cells or target prostate cancer cell-derived exosomes means that, from a heterologous mixture containing the target cancer cells, target exosomes, other cancer cells, and other cells, the target cancer cells or exosomes preferentially bind so that they are retained on the substrate and the non-target cells can be washed away or otherwise removed from the surface.

[0044] The methods and devices described herein are particularly suitable for the capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from humans. However, as will be appreciated by those in the art, target prostate cancer cells or target prostate cancer cell-derived exosomes from other organisms may be useful in animal models of disease and drug evaluation. Thus, the target prostate cancer cells or target prostate cancer cell-derived exosomes may be from other mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc.) and pets (e.g., dogs, cats, etc.).

[0045] Each cell capture surface comprises one or more prostate cancer cell or exosome selective binding agents capable of binding one or more target prostate cancer cell or target prostate cancer cell- derived exosome. The prostate cancer cell or exosome selective binding agents may be bound to the surface of a substrate by any suitable means. For example, the prostate cancer cell or exosome selective binding agents may be covalently bound directly to the cell capture surface by coupling complementary functional groups on the substrate surface and the prostate cancer cell or exosome selective binding agent. However, the prostate cancer cell or exosome selective binding agents are preferably bound to the cell capture surface via one or more intermediary layer, coating or functionality. In certain embodiments the cell capture surface of the methods and devices described herein comprises a functionalized film on the substrate. The functionalized film can comprise any inorganic, organic and/or biological material, molecule or mixture of molecules that can be attached to the surface of the substrate by covalent or ionic bonding and contain one or more functional group available for covalent or ionic bonding to prostate cancer cell or exosome selective binding agent. As described in more detail later, the prostate cancer cell or exosome selective binding agent in many cases will be a biological molecule, such as a peptide, protein or antibody and, therefore, the one or more functional group of the functionalized film is/are preferably capable of bonding to carboxylic acid groups, carboxylate groups, amino groups or amido groups on a biological molecule. [0046] Examples of chemistries suitable for covalent attachment of the prostate cancer cell or exosome selective binding agent include oxazolines, epoxies, aldehydes, anhydrides, thiols, EDC/NHS related chemistries, click chemistries, isocyanates, nitriles and imines.

[0047] In certain embodiments, the functionalized fdm is a polymer. The polymer may be formed by classical polymerization techniques. Thus, polymers can be formed on a surface of the substrate by polymerization of suitable starting monomers or pre-polymers using suitable polymerization agents, as is known in the art.

[0048] In other certain embodiments, the functionalized fdm may be formed by plasma polymerization. Thus, in certain embodiments, the functionalized fdm is a plasma polymer formed by plasma

polymerization of one or more functional starting material. Oxazoline, epoxy, aldehyde, anhydride, thiol, isocyanate, nitrile and imine containing starting materials can be plasma polymerized using the conditions described herein to form plasma polymers containing oxazoline, epoxy, aldehyde, anhydride, thiol, isocyanate, nitrile and imine groups that can then be reacted with the target prostate cancer cell selective binding agent to form covalent bonds therewith.

[0049] The conditions required to polymerise the one or more functional starting material to form the plasma polymerised functionalized fdm may comprise a power of from about 10W to about 50W, a deposition time of from about 20 seconds to about 7 minutes, and/or a monomer pressure of from about 1.1 to about 3 x 10^-1 mbar. A range of other deposition conditions would be applicable depending on the plasma deposition equipment design and power coupling efficacy.

[0050] Non-limiting examples of starting materials that can be used include 2-substituted oxazolines, 4- substituted oxazolines, 5-substituted oxazolines, 2,4-disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, 2,4,5-trisubstituted oxazolines, propionaldehyde (i.e. propanal), glycidyl methacrylate, and ally glycidyl ether.

[0051] In certain embodiments, the functionalized fdm is a plasma polymerized polyoxazoline ("PPOx"). As used herein, the term "polyoxazoline" means a plasma deposited homopolymer or copolymer formed from at least one oxazoline starting material or monomer. The polyoxazoline polymer may or may not comprise intact oxazoline moieties. The polyoxazoline polymer may be a copolymer formed by plasma polymerisation of at least one oxazoline starting material or monomer and at least one comonomer. The comonomer may be chosen based on the desired properties it may provide to the polyoxazoline polymer and/or its suitability for plasma polymerisation (e.g. its vapour pressure or volatility). The comonomer may be selected from the group consisting of, but not limited to: silanes, siloxanes, fluorocarbons, hydrocarbons, reactive functional monomers, organo-based monomers, and unsaturated monomers such as N-vinylpyrrolidone, hydroxyethylmethacrylate, acrylamide, dimethylacrylamide, dimethylaminoethylmethacrylate, acrylic acid, methacrylic acid, a vinyl substituted polyethylene or polypropylene glycol, a vinylpyridine, and a vinylsulfonic acid.

[0001 ] The plasma polymerised polyoxazoline polymer and functionalised fdm on the surface of the substrate can be prepared by exposing the surface of a substrate to a plasma comprising an oxazoline monomer vapour under conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer on the surface of the substrate.

[0052] The conditions required to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer may comprise a power of from about 10W to about 50W, a deposition time of from about 20 seconds to about 7 minutes, and/or a monomer pressure of from about 1.1 to about 3 x 10^-1 mbar. A power of greater than 30W for a time of greater than 5 minutes are particularly suitable conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer because they provide stable plasma polymerised polyoxazoline polymer films having a thickness of greater than about 5nm. However, coatings with thickness above 1 nm and deposited with a range of other conditions may be suitable too.

[0053] The plasma comprising an oxazoline monomer vapour is formed at reduced pressure in a vacuum chamber. Thus, the step of exposing the surface of a substrate to a plasma comprising an oxazoline monomer vapour may include placing the substrate in a chamber, sealing the chamber, forming a plasma in the chamber, introducing a vapour containing the oxazoline monomer into the chamber, and maintaining the substrate at a temperature suitable for polymerisation of the oxazoline monomer so as to form a polymer film on the surface. Persons skilled in the art will understand that a plasma is an electrically-excited ionised gas or gases, that, upon excitation (eg. ignition), forms a highly reactive environment that can modify materials directly exposed to the plasma discharge. The plasma deposition step can be operated over a wide range of pressures (for example, from 10 mTorr to above atmospheric pressure (eg. lOx atmosphere or higher)). The plasma may consist of a combination of an inert gas (eg. helium, neon, argon, krypton, xenon, radon) and the oxazoline monomer (or other suitable monomer as required depending on the chemistry of functionalized film). The plasma can be formed at a range of frequencies (low-frequency direct current (DC) and alternating current (AC), pulsed DC, radio frequency (RF), and microwave).

[0054] The above plasma polymerization conditions can also be used to form plasma polymers from other functional starting materials as required.

[0055] The oxazoline monomer may be a substituted oxazoline with a substituent at any of the 2-, 4- or 5-positions of the oxazoline ring or any combination of these substituents. Any of these oxazolines can be used to form the plasma polymerised polyoxazoline polymer provided they are a vapour under the plasma deposition conditions used. In embodiments, the oxazoline monomer is selected from the group consisting of 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2,4- disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, and 2,4,5- trisubstituted oxazolines. The substituent(s) on the oxazoline ring may be selected from the group consisting of: halogen, OH, NO₂, CN, NH₂, optionally substituted C₁-C₁₂alkyl, optionally substituted C₂- C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl, optionally substituted C₂-C₁₂heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted C₁-C₁₈heteroaryl, optionally substituted C₁-C₁₂alkyloxy, optionally substituted C₂-C₁₂alkenyloxy, optionally substituted C₂- C₁₂alkynyloxy, optionally substituted C₂-C₁₂heteroalkyloxy, optionally substituted C₃-C₁₂cycloalkyloxy, optionally substituted C₃-C₁₂cycloalkenyloxy, optionally substituted C₁-C₁₂heterocycloalkyloxy, optionally substituted C₂-C₁₂heterocycloalkenyloxy, optionally substituted C₆-C₁₈aryloxy, optionally substituted C₁-C₁₈heteroaryloxy, optionally substituted C₁-C₁₂alkylamino, SO₃H, SO₂NH₂, SO₂R, SONH₂, SOR, COR, COOH, COOR, CON RR^', NRCOR^', NRCOOR^', NRSO₂R^', N RCON R^'R ^", and NRR'. In some embodiments, the oxazoline monomer comprises a 2-substituted oxazoline. In specific

embodiments, the oxazoline monomer is a 2-alkyl-2-oxazoline. The alkyl substituent may be a C₁-C₁₂ alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, etc.

[0056] In specific embodiments, the oxazoline monomer is selected from the group consisting of 2- alkyl-2-oxazolines and 2-aryl-2-oxazolines. The alkyl substituent of the 2-alkyl-2-oxazolines may be a C₁-C₁₀ alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, and the like. The alkyl substituent may be optionally substituted. The aryl substituent of the 2-aryl-2-oxazolines may be a C₅-C₁₀ aryl, such as optionally substituted phenyl, optionally substituted naphthyl, optionally substituted thienyl, optionally substituted indolyl, and the like.

[0057] The surface of the substrate may be treated prior to deposition of the plasma polymerised polyoxazoline polymer. For example, the surface may be treated by cleaning with a detergent, water or a suitable solvent. Alternatively, or in addition, the surface may be treated by exposing the surface to air in a plasma chamber in order to activate the surface.

[0058] The plasma polymerised functionalized film may have a thickness of greater than 5nm, such as a thickness of about 5nm, about 6nm, about 7nm, about 8nm, about 9nm, about 10nm, about 1 1nm, about 12nm, about 13nm, about 14nm, about 15nm, about 16nm, about 17nm, about 18nm, about 19nm, about 20nm, about 30nm, about 40nm, about 50nm, about 60nm, about 70nm, about 80nm, about 90nm or about 100nm.

[0059] The reactivity of the oxazoline ring present on the w-terminus of polyoxazolines has been used for conjugation with protein and drugs in solution. The reactivity of the oxazoline ring is believed to lead to the formation of a covalent amide bond by reaction with carboxylic acid functional groups. Plasma deposited coatings allow for the retention of intact oxazoline rings at the surface, which is typically not the case when other techniques for surface preparation are used. Retention of such reactive chemical functionalities then allows convenient and rapid covalent coupling of proteins, antibodies and the like.

[0060] The prostate cancer cell or exosome selective binding agent can be any molecule that selective binds the target prostate cancer cells or target prostate cancer cell-derived exosomes. The prostate cancer cell or exosome selective binding agent may be a biomolecule. The biomolecule may for example be selected from amino acids, peptides, proteins, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents, antithrombogenic agents, and cell attachment proteins. The biomolecule may be in the form of particles or nanoparticles comprising the biomolecule.

[0061] The target prostate cancer cell selective binding agent may be attached to the functionalized fdm by direct reaction between one or more functional groups on the functionalized film and the target prostate cancer cell selective binding agent. For example, amino groups in the functionalized film may react with carboxylic, carboxylate or aldehyde groups of the prostate cancer cell or exosome selective binding agent. If required, reaction between the one or more functional group on the functionalized film and the target prostate cancer cell selective binding agent may be facilitated or catalyzed. For example, reaction between the one or more functional group on the functionalized film and the prostate cancer cell or exosome selective binding agent may be facilitated by a coupling agent such as a carbodiimide coupling agent (for example EDC, DCC, DIC), a triaminophosphonium coupling agent (for example BOP, PyBOP, PyBrOP) or a tetramethylaminium/tetramethyluronium coupling agent (for example HATU, HBTU, HCTU).

[0062] A cross linker moiety may be used between the functionalized film and the prostate cancer cell or exosome selective binding agent.

[0063] The prostate cancer cell or exosome selective binding agent can be any inorganic, organic and/or biomolecule that allows the device to selectively capture the target prostate cancer cells or target prostate cancer cell-derived exosomes. The prostate cancer cell or exosome selective binding agent may comprise at least two functional groups. A first functional group of the prostate cancer cell or exosome selective binding agent is a moiety capable of attaching to one or more functional group of the functionalise film, as described earlier.

[0064] Selection of the appropriate prostate cancer cell or exosome selective binding agent is a matter of choice that depends upon the particular target prostate cancer cell or target prostate cancer cell-derived exosome population of interest. [0065] A second functional group of the prostate cancer cell or exosome selective binding agent is capable of binding to a cell or exosome. The binding between the second functional group and the cell or exosome can be direct or indirect. For binding directly to a cell or exosome, the second functional group itself comprises an active group capable of recognising and capturing a cell or exosome. The active groups can be specifically selected to recognise and capture a specific cell or exosome type of interest, and cell recognition and capture can be accomplished by any means known in the art. For example, cell recognition can be based on chemical or biological reactions, including without limitation peptide recognition, nucleic acid recognition and/or chemical recognition. Cell recognition can also be based on non-chemical or non-biological reaction, such as, without limitation, electrokinetic recognition or size- dependent sorting.

[0066] To indirectly bind to a cell or exosome, the second functional group of the prostate cancer cell or exosome selective binding agent is attached to a cell or exosome through a separate cell-binding agent. Such cell-binding agents are well-known in the art. The cell-binding agent may be a single component or be in a form of complex comprising two or more components, as long as at least one of the components is capable of binding to a target cell or target cell-derived exosome. For example, in addition to the component directly binding to a cell or exosome, the cell-binding agent or complex may comprise an additional component attached to the component binding to the cell or exosome.

[0067] For example, the prostate cancer cell or exosome selective binding agent, or one component of the prostate cancer cell or exosome selective binding agent, can be an antibody, a lymphokine, a hormone, a growth factor, or any other cell-binding molecule or substance that specifically binds a target prostate cancer cell or target prostate cancer cell-derived exosome.

[0068] In certain embodiments, the prostate cancer cell or exosome selective binding agent is one or more antibodies, or fragments thereof. The antibodies may be selected from: polyclonal antibodies or monoclonal antibodies, including fully human antibodies; single chain antibodies (polyclonal and monoclonal); fragments of antibodies (polyclonal and monoclonal) such as Fab, Fab', F(ab')₂, and Fv; chimeric antibodies and antigen-binding fragments thereof; and domain antibodies (dAbs) and antigen- binding fragments thereof, including camelid antibodies. In certain specific embodiments, the prostate cancer cell or exosome selective binding agent is a monoclonal antibody. In other certain specific embodiments, the prostate cancer cell or exosome selective binding agent is a polyclonal antibody.

[0069] Monoclonal antibody techniques allow for the production of specific cell-binding agents in the form of monoclonal antibodies. Techniques for creating monoclonal antibodies are well known in the art. Such antibodies can be produced by, for example, immunizing mice, rats, hamsters or any other mammal with the antigen of interest. Antigens of interest may include the intact target prostate cancer cell, antigens isolated from the target prostate cancer cell, whole virus, attenuated whole virus, or viral proteins such as viral coat proteins. Sensitized human cells can also be used. Another method of creating monoclonal antibodies is the use of phage libraries of scFv (single chain variable region), specifically human scFv.

[0070] The prostate cancer cell or exosome selective binding agent can also be a combination of two or more different kind antibodies.

[0071] Thus, the methods and devices described herein may comprise an immobilized functional antibody capable of selective capture of target prostate cancer cells or target prostate cancer cell-derived exosomes. The present inventors have found that anti-prostate specific membrane antigen (PSMA) antibodies in particular are able to selectively capture prostate cancer cells from urine and urine derived fluids on the substrate surface. Therefore, in certain embodiments the immobilized functional antibody is an anti-PSMA antibody. PSMA is a type II transmembrane zinc metallopeptidase tumor marker in prostate cancer encoded by the FOLH1 gene. Prostate cancer cells express significantly more PSMA than other tissues like kidney, salivary glands and proximal small intestine (Afshar-Oromieh et al., 2013; Chang et al., 1999; Eder, Eisenhut, Babich, & Haberkom, 2013; Eder et al., 2012; Hillier et al., 2009; O'Keefe, Bacich, & Heston, 2004). PSMA is expressed about 8 to 12 fold more in some prostate cancer cells in comparison to non-cancerous prostate cells. This elevated expression of PSMA makes it a promising biomarker for cancer therapy and imaging (O'Keefe et al., 2004; Wang et al., 2007). Yet, to the best of the inventors' knowledge, this membrane protein has not yet been used for the selective capture of whole prostate cancer cells from urine samples. In certain specific embodiments, the antibody is an anti- PSMA antibody. A number of anti-PSMA antibodies are commercially available and/or details are provided in the literature and the person skilled in the art can readily determine the applicability of any of the known anti-PSMA antibodies by following the methods provided in the Examples section of this specification. In certain embodiments, the anti-PSMA antibody is selected from the group consisting of human PSMA/FOLH1 phycoerythrin MAb (Clone 107-1A4, see Brown et al., Prostate Cancer Prostatic Dis. 1998 Jun;1(4):208-215; also commercially available from R&D Systems, Inc. Minneapolis, MN 55413) (hereafter“107-1A4”) and anti-PSMA antibody [GCP-05] ab66912 (commercially available from Abeam, Cambridge, MA, USA) (hereafter“ab66912”). In certain specific embodiments, the anti-PSMA antibody is 107-1A4. Other anti-PSMA antibodies are available and can be used.

[0072] One important challenge is for the antibody to remain strongly bound to the cell capture surface despite high variability in pH and ionic concentration in urine samples. This challenge can be overcome by using the polyoxazoline plasma polymers to covalently bind the antibody to the substrate.

[0073] Non-antibody molecules can also be used to target specific target prostate cancer cell or target prostate cancer cell-derived exosome populations. [0074] Once captured on the cell capture surface of the substrate, target prostate cancer cells or target prostate cancer cell-derived exosomes can be separated from urine and other components of urine by washing, for example with PBS.

[0075] Captured target prostate cancer cells or target prostate cancer cell-derived exosomes can be detected and/or analysed using any of several methods known to those skilled in the art. Captured target prostate cancer cells or target prostate cancer cell-derived exosomes can be observed using a

photomicroscope. Captured target prostate cancer cells or target prostate cancer cell-derived exosomes may be detected by fluorescent or luminescent labelling. For example, captured target prostate cancer cells or target prostate cancer cell-derived exosomes can be imaged using fluorescent microscopy. The number of cells or target prostate cancer cell-derived exosomes bound to the surface can then be computed.

[0076] Advantageously, captured prostate cancer cells can be differentiated in vitro from healthy cells by using a cancer specific fluorescently active compound. For example, captured prostate cancer cells can be detected using a cancer specific photosensitiser composition. Cancer specific photosensitiser compositions that can be used to detect captured prostate cancer cells include, but are not limited to, compositions comprising 5-aminolevulinic acid (5-ALA), hexaminolevulinate (HAL) and/or hypericin.

[0077] 5-ALA is a natural amino acid synthesized from succinyl-CoA and glycine in haem

biosynthesis. 5-ALA does not fluoresce, but is a precursor of the natural photosensitizing metabolite, protoporphyrin IX (PpIX). Exogenous administration of 5-ALA increases the accumulation of PpIX primarily in tumour tissues. When excited by blue light, tumour cells emit a bright red fluorescence, reportedly several fold brighter than normal healthy cells.

[0078] Hexaminolevulinate (HAL) has been successfully used as a 5-ALA derivative. It has been reported to increase PpIX fluorescence intensity in lower concentrations and with shorter exposure than 5-ALA itself (Lange, N., et al. 1999; Abdel-Kader, 2014). HAL is more lipophilic than 5-ALA, which enhances the cellular uptake through the plasma membrane.

[0079] The cancer specific photosensitiser composition is combined with a sample of urine or a urine derived fluid. In these methods, the cells are in suspension in the urine or urine derived fluid sample and are not bound to a surface. As such, the cells are free and this provides maximum exposure of the cells to the cancer specific photosensitiser composition before diagnosis.

[0080] In certain embodiments, the captured prostate cancer cells can additionally be detected using a secondary luminescent cell nucleus stain composition. This can advantageously provide improved classification of cells containing a nucleus. In certain embodiments, the luminescent cell nucleus stain is fluorescent. In certain embodiments, the luminescent cell nucleus stain is Nuclear-Red stain (ie. 4-amino- 9,10-dihydro-1,3-dihydroxy-9,10-dioxo-2-anthracenesulfonic acid sodium salt or kemechtrot) or Nuclear Blue stain (ie. Hoechst 33342 or 2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H- benzimidazole trihydrochloride trihydrate).

[0081] In certain embodiments, the luminescent cell nucleus stain composition comprises a cell penetrative solvent. In these or other embodiments, the luminescent cell nucleus stain composition comprises an iron chelating solvent. In specific embodiments, the luminescent cell nucleus stain comprises dimethyl sulfoxide (DMSO).

[0082] The concentration of luminescent cell nucleus stain in the luminescent cell nucleus stain composition may be from about 0.1 mM to about 10 mM. As will be appreciated, the concentration of luminescent cell nucleus stain used will depend on the nature of the stain used. For example, the concentration of Nuclear Blue stain may be about 10 mM. The concentration of Nuclear-Red stain may be from about 0.1 mM to about 1 mM, such as about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM or about 1.0 mM. In certain embodiments, the concentration of luminescent cell nucleus stain in the luminescent cell nucleus stain composition is about 0.5uM.

[0083] If desired, captured target prostate cancer cells can be selectively released from the capture surface of the substrate. This then allows for subsequent genetic analysis of cells if required.

[0084] Alternatively, or in addition, in certain embodiments captured prostate cancer cells or target prostate cancer cell-derived exosomes can be detected by using other techniques known in the art including, but not limited to, antibody staining and genetic analysis. For example, the genetic content of the captured prostate cancer cells or target prostate cancer cell-derived exosomes can be tested using PCR to detect specific micro RNAs and DNA fragments of prognostic value. By way of example, following the method of Tinzl, an amplification assay (uPM3™) can be used to detect DD3^PCA3 RNA in captured prostate cancer cells or prostate cancer cell-derived exosomes (Tinzl et al., 2004).

[0085] It will be apparent from the foregoing disclosure that the present disclosure provides a method for diagnosing or monitoring prostate cancer in a mammal. The method comprises providing a sample of urine or a urine derived fluid obtained from the mammal; providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; contacting the sample of urine or a urine derived fluid with the one or more cell capture surfacs under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell-derived exosomes from the urine (if present) to the cell capture surface; and detecting any target prostate cancer cells or target prostate cancer cell-derived exosomes bound to the cell capture surface.

[0086] It will also be apparent from the foregoing discussion that the methods described herein can be used to capture or immobilise prostate cancer cells or prostate cancer cell-derived exosomes on a surface. Thus, the present disclosure also provides a method of immobilising target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid on a substrate surface. The method comprises providing a sample of urine or a urine derived fluid; providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; and contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell-derived exosomes from the urine (if present) to the cell capture surface.

[0087] The devices and methods described herein provide a rapid and selective method for capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from urine. Current urinary diagnostic tests for prostate cancer are expensive and have limited sensitivity and specificity. The devices and methods described herein provide the first generation of specific urinary tests for the detection of prostate cancer cells or prostate cancer cell-derived exosomes in urine.

[0088] The devices and methods described herein combine the attributes of anti-PSMA antibodies and fluorescence detection without any side effects for the patients. The devices and methods can be used for detecting prostate cancer higher sensistivity and/or specificity than presently available methods used in prostate cancer detection.

EXAMPLES

[0089] Materials

[0090] Hexaminolevulinate (HAL) hydrochloride was purchased from Sigma-Aldrich (NSW,

Australia). HAL was supplied as a powder and was dissolved in phosphate -buffered saline (PBS).

Solutions of different concentrations (from 0 to 150mM) were prepared. Nuclear-Red™ LCS1 (Cat# 17542) which is a cell-permeant nucleic acid detection dye was obtained from AAT Bioquest® (CA 94085, USA). Chemicals were stored in aliquots at -20°C until use. DAPI from Sigma Aldrich Australia was used as received and stored at -4°C. Dimethylsulphoxide (DMSO) was purchase from Sigma-Aldrich (NSW, Australia) and used as the main solvent for Nuclear-Red dye. [0091] Plasma polymerization

[0092] Prior to the plasma deposition, solid substrates (microscopy grade-glass slides, silicon wafers and plastic coverslips) were washed with acetone and ethanol for 10 min each in an ultrasonic bath. A custom-fitted parallel plate plasma reactor placed under a base pressure vacuum (2.0e^-2 mbar) was used to deposit an oxazoline thin film coating on the substrates as previously described (Macgregor-Ramiasa, McNicholas, Ostrikov, Li, Michael, Gleadle, Vasilev, et al., 2017). Briefly, a needle valve enabled the introduction of a 1, 7-octadiene (Sigma Aldrich Australia) precursor into the chamber after reaching a 1.3e^-1 mbar working pressure. A continuous radiofrequency power of 20W ignited the plasma for 1 minute to form a polyoctadiene (POD) under-layer on the substrate ensuring the stability of the polyoxazoline (POX) thin film on the surface. The upper layer was deposited from a 2 -methyl-2 - oxazoline (Sigma Aldrich Australia) precursor inserted with a 1.3e^-1 mbar flowrate for 3 minutes with a 50 W RF power. Polymethylmethacrylate (PMMA) industrially made microchannel slides were also used directly for the PPOx-film deposition without any modification (cleaning or PPOD under-layer deposition). The coated slides were kept under vacuum between 24h to one week after the deposition in readiness for a further characterization or functionalization (MacGregor et al). The assembled POX coated chips consist of three microchannels (18 mm × 4 mm × 500 mm). The base of the chip is made of PMMA and features the three channel grooves coated with plasma deposited POX. The top part of the chips feature the fluid inlets and outlets for each channel. The chip is assembled by sticking the base and top PMMA parts together with adhesive.

[0093] A functionalized oxazoline film was also formed using 2-isopropenyl-2-oxazoline precursor (Figure 1). Advantageously, it was found that this precursor works accross a wider range of deposition conditions (10-50W RF power, 1 to 1.3e^-1 mbar) when compared to 2-methyl-2 -oxazoline. The resultant functionalized film was more stable so it didn't need an octadiene underlayer. The 2-isopropenyl-2- oxazoline based functionalized film (PiPOx) was formed on glass slides and PMMA microchannels.

[0094] Functionalization of the polyoxazoline (POX) coated substrate

[0095] The POX coated chips were functionalized with Anti-PSMA antibodies (PSMA) and skim milk solution (Block). Negative and positive controls were needed as a comparison with PSMA functionalized channels to approve the selective cancer cell capture (Fujita et al., 2009b). The positive controls were made of the unmodified PPOX coated surface, allowing a non-specific cell capture and the negative control consisted of PPOX coating blocked with skim milk proteins. PSMA were diluted in a 100% PBS solution to reach different concentrations of 10, 25, 50 and 75 mg/mL , which was then pipetted into the coated microchannels and stored overnight at 4°C to ensure the irreversible binding between the antibodies' carboxylic acid function and the oxazoline-based surface (Pal et al., 2016b). Once the PSMA solution was removed, microchannels were incubated for an hour at 37°C, 120 rpm in 1mg.mL^-1 skim milk solution, in order to avoid any non-specific cell binding (Nickens et al., 2015b). The chip was finally rinsed three times with fresh PBS solution (Figure 2).

[0096] Microchannels were also functionalised with Anti-PSMA antibodies (PSMA) for patient urine sample tests using the same procedure. The PSMA concentration was 10ug/mL.

[0097] Cell culture

[0098] Human Caucasian prostate carcinoma cell line LNCaP (ECACC No. 89110211) and human prostate normal cell line PNT2 (ECACC No. 95012613) were obtained from European Collection of Authenticated Cell Cultures (ECACC). LNCaP and PNT2 cells were cultured in RPMI 1640 medium (Life Technologies, Australia) supplemented with 10% (v/v) fetal calf serum, ImM sodium pyruvate,

100 IU/mL penicillin, 1% L-glutamine (200mM) and 100 mg/mL streptomycin. All cells were cultured at 37°C with 5% CO₂ in a humidified atmosphere.

[0099] Cell preparation for cancer specific fluorescence investigation

[00100] LNCaP and PNT2 cells were trypsinized from the culturing flask, resuspended in the serum-free medium to dilute the trypsin, and adjusted to the targeted cell density. The cell suspensions were incubated at different time points with different concentrations of HAL (0, 50, 100 and 150mM) in PBS at temperatures of 37°C, 23°C and 4°C. The cells were kept in the dark for the entire HAL incubation period. Moreover, cells at 37°C were treated with Nuclear-Red stain for the last 10 minutes of the 1-hour HAL incubation. After incubation, no centrifugation took place. Prior to imaging, 100 ml of cells were added per well in 96 well plates and imaged using fluorescence microscopy. The recorded images were analysed using CellSens software. The mean value and standard deviation (SD) of fluorescence intensities were calculated. The significance differences were analyzed by student's t-test (GraphPad software). Statistical significance was considered as p<0.05.

[00101] Cell preparation selective whole cell capture

[00102] Iteratively challenging experiments were designed to investigate the selective capture of LNCaP cells. Firstly, LNCaP and PNT2 cells were stained with Nuclear-Red (0.5-mmol.L-¹) solution and DAPI for 10 minutes respectively, in separate tubes to eliminate possible stain crossover. The cell suspensions were then centrifuged at 5000 rpm for 5 min, the supernatant removed and the cell pellet resuspended in fresh PBS. In a second iteration, real urine collected from a healthy donor was used as media rather than PBS. The specimen was centrifuged for 5 minutes at 1500 rpm. The supernatant was collected and filtered twice through a 0.2 mm pore syringe filter, and used as the urine media. LNCaP and PNT2 cells were stained as in the first experiment with Nuclear-Red and DAP1. 50 mL of stained cell suspension was added to 950 mL of urine media to make 1 mL. The spiked urine cell sample was then immediately used.

[00103] In the final experiments, instead of using DAPI and Nuclear-Red dye to discriminate between healthy and cancer cells captured, the feasibility of using HAL cancer specific fluorescence for diagnostic purposes was tested. Hence, three different lots of cells were prepared: A) PNT2 with no stain, B) PNT2 with 50 mmol.L^-1 HAL and C) LNCaP with 50 mmol.L^-1 HAL and Nuclear-Red (0.5-mmol.L-¹) and mixed with PNT2 plus 50 mmol.L^-1 HAL. For all HAL, incubation time was 1 hour at room temperature in the dark and for‘C’ Nuclear-Red was added after the HAL incubation time.

[00104] In all the experiments, functionalised test channels were loaded with the mixture of stained cells. The test chip was imaged with a fluorescence microscope. After 45 minutes, the capture channels were rinsed with PBS to dislodge any unbound cells and imaged again to observe captured cells on the surface. After the experiments, the number of cells in each channel before and after wash was counted in each channel to calculate the capture sensitivity and specificity. The results are expressed as per the equations below:

[00105] Patient sample processing

[00106] For a clinical study, urine specimens were prospectively collected from 10 men admitted to the urology department of Flinders Medical Centre in accordance with protocol approved by the Southern Adelaide Clinical Human Research Ethics Committee. The patient cohort consisted of three groups: two patients with hystopathologically confirmed prostate cancer, two patients with benign prostate hyperplasia - also typically associated with an elevated PSA level (Kehinde et al., 2003), and six control patients admitted to the urology department for conditions other than cancer. Each patient provided a written consent prior to collection of the urine sample for this study. Table 1 presents some information concerning the patients in this study. It shows some baseline characteristics of the patient cohort.

[00107] 50 mL of urine was collected from each patient admitted in the trial. The specimens were transported and kept at 4°C until further processing which occurred within 4 hours post collection. Once received, the samples were agitated gently to resuspend any settled material, and 5 mL of urine was then incubated under the conditions identified in the final cell line experiments, namely 1 hour at room temperature in the dark with HAL at 50 mmol.L^-1. After settling patient samples for 1 hour of HAL incubation, 300 mL of the settled urine was collected and Nuclear-Red at 0.5mmol.L^-1 was added. The test chip was then loaded with the stained urine sample and ready to image with the fluorescence microscope.

[00108] PSMA capture from urine samples was also performed in the same way.

[00109] Table 1 - Study participant patient backgrounds

[00110] Fluorescence Microscopy Imaging

[00111] Images were acquired using a fluorescence microscope with objective lenses of 4, 5 and 10 X. The fluorescence microscope contained different filters set for imaging DAPI, Nuclear-RedTM, HAL- induced PpIX fluorescence and bright filed. The standard DAPI filter cube used a 350/50 nm excitation filter and 460/50 nm emission filter. Nuclear-Red was imaged through a standard Cy5 filter set with a 620/60 nm excitation filter and a 700/75 nm emission filter. PpIX fluorescence was imaged through a custom-made filter cube set with excitation at 405/20 nm and emission through a long pass 610 nm emission filter.

[00112] Results and Discussion

[00113] HAL concentration, incubation time and temperature effect on cancer specific fluorescence

[00114] To determine the feasibility of using HAL-induced PpIX fluorescence for human prostate cancer and non-cancer cell lines identification, the effect of different environmental factors on HAL-mediated PDD was tested. Cells were incubated with various HAL concentrations (50, 100, and 150mM), for different periods (0.5h, 1h and 2h), and temperatures (4°C, 23°C and 37°C) in triplicate.

[00115] Prostate cancer LNCaP cells showed higher fluorescence intensities than non-cancer PNT2 cells when incubated with HAL. Figure indicates that the accumulation of PpIX increased significantly in a time and temperature-dependent pattern for LNCaP cells when the cells were incubated at 37°C and 23°C. In contrast, only a very slight increase was observed at 4°C. In addition, the results suggested that the cancer specific PpIX fluorescence is a dose-independent process in the cell suspensions investigated. No significant difference was observed in the fluorescence intensity of non-cancer PNT2 cells treated with HAL for all temperature settings after 0.5h and 1h. At 2h time point, PNT2 cells display a small, yet non-significant increase in PpIX fluorescence when incubated at 37°C and 23°C.

[00116] Overall, higher PpIX fluorescence intensities were displayed in cancer LNCaP cells upon exposure to HAL especially when incubated at 37°C: Most notably, a significant 1 and 2 folds increase in fluorescence was observed in LNCaP cells compared to PNT2 cells, for 1 and 2 h at 37°C, respectively.

5 -ALA is a precursor for the heme biosynthetic pathway and production of PpIX. Heme biosynthesis is controlled by the existence of various enzymes. In the previous studies, the PpIX production increases with temperature in human skin cells (Kim, Koo, Kim, & Kim, 2016; Mamalis, Koo, Sckisel, Siegel, & Jagdeo, 2016). Our results, showing higher PpIX production at 37°C than 23°C and 4°C. It further indicates that the cancer specific HAL induced fluorescence is a process that seems to occur when intracellular enzymes are active. In other words, the fluorescence may not be as pronounced in fixed or dead cells when there is very little or none uptake of HAL and/or enzymatic activity happened. For practical purposes, our results indicate that the optimal HAL incubation condition is 50 mM at 37°C for 1 to 2 hours.

[00117] Nuclear stain effect on HAL-induced cancer specific fluorescence

[001 18] For real life application of the device, staining of cell nucleus is necessary to discriminate between cells and other urinary artefacts that may exhibit autofluorescence in the HAL optical channel. It is therefore important to test whether or not the combination of Nuclear-Red and HAL does not have a detrimental effect on the cancer specific fluorescence in the optimal HAL-incubation conditions identified above. The differences of the PpIX fluorescence between prostate cancer LNCaP and non-cancer PNT2 cells after 1 hour HAL incubation in the presence of Nuclear-Red are shown in Figure Figure 9-a. The presence of Nuclear-Red had an effect on the fluorescence intensity of LNCaP cells but not on that of the healthy PNT2 cells. Nor did PNT2 show any differences in fluorescence intensity with and without Nuclear-Red treatment. In LNCaP cells, HAL induced PpIX fluorescence upon the incubation with Nuclear-Red was initially elevated in 50 and 100mM HAL. The impact of NuclearRed incubation duration on the cellular PpIX fluorescence was also investigated. The results show that there is no difference between the cells treated with Nuclear-Red for the full 1 hour or only for the last 10 minutes of the HAL incubation time. The percentage of PpIX fluorescence in the LNCaP counts was higher across 50 and 100mM HAL with NuclearRed. In the case of PNT2, the reverse trend was observed, namely the healthy cell fluorescence was slightly less in the presence of the nuclear stain, though the difference was not significant (Figure 3-B).

[001 19] The main solvent of NuclearRed dye, dimethyl sulphoxide (DMSO), may be the reason for the elevated PpIX fluorescence intensities in cancer cells incubated with the nuclear stain. Indeed, it has been shown that DMSO is a penetration enhancer and an iron chelator (Fujita et al., 2009b; D. Hessels & J. A.

J. A. j. o. a. Schalken, 2013; Nickens et al., 2015b; Pal et al., 2016b), two aspects that may favour PpIX accumulation in cancer cells. Malik et al. reported that DMSO could promote the PpIX accumulation in colon tumour cells by 1) increasing HAL cell membrane penetration; and 2) chelating iron thus hindering the process by which PpIX is turned into the next compound in the heme synthesis pathway. This is related to multiple heme biosynthesis enzymes and porphyrin transporters alterations in tumour cells when compared with normal cells. Ferrochelatase (FECH) is the enzyme converting PpIX to heme in the mitochondria, lower FECH expression has been found in colorectal (Bogdanov Jr, 2008) and bladder cancer cells compared to normal cells (Krieg, Fickweiler, Wolfbeis, Knuechel, & photobiology, 2000). Silencing of FECH by iron chelators increases the PpIX accumulation in prostate cancer cells (Elsevier, 2009). Porphyrin transporters also play an important role in PpIX metabolism. ATP-binding cassette sub- family G member 2 (ABCG2) mediate the efflux of PpIX from mitochondria to cytosol (Kobuchi et al., 2012), suppressed ABCG2 increases the PpIX accumulation in several cancer cell lines including histiocytic lymphoma, colorectal and bladder (Hagiya et al., 2013; Kobuchi et al., 2012). While DMSO has been linked to the induction of cell differentiation and enzymatic activation in the heme biosynthesis, further investigation is necessary, to gauge the potential of DMSO as an enhancing agent of HAL- induced fluorescence in cancer cells (Malik et al., 1995).

[00120] Selective cancer cell immunocapture

[00121] In previous work we used anti-EpCAM antibody for the selective capture of carcinoma cells - which overexpress EpCAM compared to healthy epithelial cells- from urine samples in microfluidic devices (Macgregor-Ramiasa, McNicholas, Ostrikov, Li, Michael, Gleadle, & Vasilev, 2017). PSMA is a promising prostate cancer specific biomarker located on the membrane of the tumor cells (O'Keefe,

Bacich et al. 2004, Wang, Yin et al. 2007). This makes anti-PSMA a worthy candidate for the selective immunocapture of prostate cancer cells following the microfluidic-based whole cell capture approach we developed earlier. Western blot analysis was performed on both healthy prostate PNT2 and cancerous LNCaP cells using antibodies to PSMA and EpCAM. A bladder cancer cell line (RT4 was used as positive control for EpCAM expression) Figure 5. a., shows that the two prostate epithelial cell lines do express EpCAM, but only the malignant LNCaP cells expressed PSMA in detectable levels, thus confirming that PSMA is a suitable membrane marker to distinguish a healthy from a cancerous prostate cell type.

[00122] Different antibodies (107-1A4 and ab66912) were therefore tested for the selective capture of LNCaP cells. The positive control surface, unmodified PPOX, showed 104% and 99% cell capture for both LNCaP and PNT2 cell lines, respectively. The negative control surface, skim milk blocked, consistently captured less than 3% of both cell types. The capture rate for PNT2 and LNCaP cell lines on modified functionalised surfaces (107-1A4 at 10, 25, 50 and 75 mg/ml and ab66912 at 20 mg/ml) is shown in Figure 4.C. For PSMA antibody 107-1A4, at 10 mg/ml, the capture rate was 97% which is the highest capture rate amongst all others. However, 25 and 50 mg/ml has also reach a satisfactory capture rate of 90% which may be deemed acceptable for practical application. At 75 mg/ml, the rate decreased to 75%. At this concentration, the surface bound antibody may start to form multi-layers that would decrease the number of available binding sites for the cells to be captured. The ab66912 at 20 mg/ml showed less capture, namely about 79%, in comparison to the 107-1A4 at 10 mg/ml. This indicated that 107-1A4 is better suited for the selective capture of prostate cancer cells. Overall, as the best condition for capturing LNCaP cells in the future is to use the 107-1A4 at 10 mg/ml, which gives the best specificity (93.7%) and sensitivity (97.9%).

[00123] Selective cancer cell immunocapture in urine media

[00124] The selective capture experiment was repeated using urine as media for the cell suspension and compared to PBS media. Figure Figure 5 shows that unmodified PPOX as a positive control captured 100% of LNCaP and 94.50% of PNT2 cell in PBS media. Similar capture rates were obtained for both cell lines in urine media where 97.94% and 93.21% of cells were capture for LNCaP and PNT2 respectively. The block channel as a negative control surface captured less than 8% of both cell types in both PBS and urine media. The capture rates for LNCaP cell lines on modified antiPSMA-functionalised surfaces were 92% in PBS media and 89% in urine media, while for healthy PNT2 cells they were below 8% in both media. This data indicates that using the 107-1A4 at 10mg/ml in both urine and PBS media led to approximate sensitivity of 90% and specificity of 95%. Hence, it is verifying one more time that, for capturing LNCaP cells in the future, PSMA 107-1A4 at 10 mg/ml could be used as the best option.

[00125] Selective capture cancer cells immunocapture with cancer specific HAL-induced fluorescence

[00126] In a final experiment. LNCaP and PNT2 cells were both incubated with HAL to evaluate the capability of the cancer specific HAL-induced fluorescence to discriminate between healthy and cancer cells in the cell population captured on the PSMA functionalised microchannel. In this experimental setting, LNCaP cell -only- were also stained with Nuclear-Red, as a control measure. It can be seen from Figure 6 that no PNT2 cells in both‘A’ (not stained) and‘B’ (stained with HAL), were captured in the PSMA-A and PSMA-B surfaces. On the other hand, PSMA-C showed more than 80% capture in both Nuclear-Red and HAL channels which indicates the selectivity of the chip in capturing the prostate cancer cells. Additionally, the percentage of cell capture in bright field of PSMA-C (44.4%) shows that almost half of the cells that were PNT2 washed off the surface and only LNCaP cells were captured. Figure 7 indicates that LNCaP cells express HAL induced PplX fluorescence where as PNT2 cells not. Hence, combining HAL and Anti-PSMA (107-1A4 at 10mg/ml) provide an approach for the detection of prostate cancer cells in a mixed cell population with a dual confidence level: 1) the selective immuno-capture based on PSMA overexpression; and 2) the PplX fluorescence based on cancer specific HAL-induced fluorescence.

[00127] Selective cancer cell capture from patient samples

[00128] A preliminary study has been done with prostate patient samples to indicate the ability of the current microchannel test chip in capturing prostate cancer cells without massaging the prostate itself that has been mentioned as one of the ways to release the cancer cells in urine (Nakai et al., 2018; Nickens et ah, 2015b). 2 prostate cancer patient samples and 2 benign prostate conditions (hyperplasia) together with 6 control patient samples were tested in duplicates. HAL absolute cell number in EpCAM channel was counted and compared to the clinical diagnosis (cystoscopy). The results obtained on this small patient cohort have shown the highest possible sensitivity, specificity, PPV and NPV (100%) (Table 2). This pilot data proves that prostate cancer could be detected through the current defined test chip without massaging the prostate and with non-processed patient urine samples. In addition, PCR tests were conducted on the cellular material contained in 1 and 5mL urine samples from patients diagnosed with prostate cancer or control patients with benign prostatic conditions. These tests confirmed the presence of cells expressing AR and KLK3 genes, which confirmed the presence of cells of prostatic origin. In confirmed PCA patient samples, PCR also confirmed the presence of cells expressing PSMA and/or PCA3, indicating there are malignant cells present in the urine.

[00129] Our further data also shows that using the PiPOx slide described in paragraph [0092] functionalised with anti-PSMA as described in paragraph [0095], selective capture of PplX positive cells was achieved from urine samples of confirmed prostate cancer patients, tested following the procedure described in paragraph [00107] (n=17). [00130] Table 2 - Screening patient samples with the test chip using HAL absolute cell number in EpCAM channel and compared to the clinical diagnosis.

[00131] It has been shown that even by using another type of cancer specific antibody, some prostate cancer cells were captured using the current microfluidic test chip. This suggests that by using PSMA antibody more, prostate cancer cells could be captured.

[00132] Prostate cancer cells selective capture with Olympus microscope

[00133] Parallel experiments have been conducted using an Olympus microscope in where 107-1A4 used at 10 mg/ml. which has been shown to be the best concentration with 97% capture rate. Similar results have been observed with 96% capture for the 107-1A4 and 72% for ab66912. The unmodified PPOX performed as expected which showed 98% capture rate for LNCaP and 105% for PNT2 cells. EpCAM has also been used as the control as both LNCaP and PNT2 cells are expressing EpCAM that could be clearly observed in Figure 8.

[00134] Histogram data obtained from the PpIX fluorescence images

[00135] Histogram data obtained from the PpIX fluorescence images of different ratios of cancer and non-cancer cells mixed are shown in Figure 9. Cells were incubated with 50mM HAL in 37°C for 1hour and 2 hours respectively. The entire fluorescence data were collected and analyzed using software developed by SMR Automotive

[00136] Conclusion

[00137] The number of people with prostate cancer is increasing every single day. Current methods for diagnosis have some limitations such as inaccuracy, invasiveness, and not being selective and/or specific enough. A new cancer diagnostic platform, microchannel test chip, has been investigated in this study, which is non-invasive. Previous research conducted by independent groups, has shown that PSMA and HAL could be used to identify prostate cancer cells (Abdallah & Kassem, 2007; Afshar-Oromieh et al., 2013; Eder et al., 2013; Fotinos et al., 2008; Jichlinski et al., 2003; Nakai et al., 2017; Nitzan et al., 2004). Here, the combination of the two has been used in a newly developed microfluidic chip to provide better sensitivity and specificity towards prostate cancer diagnosis and prognosis.

[00138] Detailed optimisation of HAL concentration, incubation time and temperature indicated that the best discrimination between healthy and cancer cells was obtained for 50-100uM HAL incubated with the cells for 1 hour at 37°C.

[00139] The addition of a nuclear stain, which is necessary in the practical application of the diagnostic test, did not impair the specificity of HAL fluorescence. In fact, adding Nuclear-Red stain increased the difference in fluorescence intensity between healthy and cancer cells. Several antibodies and

concentrations were tested for the selective capture of prostate cancer cells in a newly developed microfluidic chip to define optimal capture conditions.

[00140] Anti-PSMA 107-1 A4 at 10 mg/ml led to the best specificity (93.7%) and sensitivity (97.9%). In a final assay, this antibody functionalisation was used in combination with assay HAL and Nuclear-Red staining to validate the feasibility of the dual-method for the selective detection of prostate cancer cells. Using this combined approach, sensitivity and specificity of 86.2% and 88.1% respectively were achieved. This new designed device with grouping immune capture antibody and cancer specific photosensitizers could be applied to capture prostate cancer cells with better sensitivity and specificity than the current blood test, which is the gold standard.

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[00184] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00185] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

Claims

1. A method of selective capturing target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid, the method comprising:

providing a sample of urine or a urine derived fluid;

providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; and

contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell- derived exosomes from the urine (if present) to the cell capture surface.

2. The method according to claim 1, wherein the prostate cancer cell or exosome selective binding agents comprise one or more PSMA antibody or functional equivalent thereof.

3. The method according to either claim 1 or claim 2 and further comprising detecting target prostate cancer cells or target prostate cancer cell-derived exosomes on the one or more cell capture surface.

4. The method according to claim 3 comprising detecting captured prostate cancer cells using a cancer specific photosensitiser composition.

5. The method according to claim 4, wherein the cancer specific photosensitiser composition is selected from the group consisting of compositions comprising 5-aminolevulinic acid (ALA 5), compositions comprising hexaminolevulinate (HAL), and compositions comprising hypericin.

6. The method according to any one of claims 3 to 5 further comprising detecting the captured prostate cancer cells using a luminescent cell nucleus stain composition.

7. The method according to claim 6, wherein the luminescent cell nucleus stain is Nuclear-Red stain.

8. A method for diagnosing or monitoring prostate cancer in a mammal, the method comprising: providing a sample of urine or a urine derived fluid obtained from the mammal;

providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell- derived exosomes from the urine (if present) to the cell capture surface; and

detecting any target prostate cancer cells or target prostate cancer cell-derived exosomes bound to the cell capture surface.

9. The method according to claim 8, wherein the prostate cancer cell or exosome selective binding agents comprise one or more PSMA antibody or functional equivalent thereof.

10. The method according to either claim 8 or claim 9 comprising detecting captured prostate cancer cells using a cancer specific photosensitiser composition.

1 1. The method according to claim 10, wherein the cancer specific photosensitiser composition is selected from the group consisting of compositions comprising 5-aminolevulinic acid (ALA 5), compositions comprising hexaminolevulinate (HAL), and compositions comprising hypericin.

12. The method according to any one of claims 8 to 1 1 further comprising detecting the captured prostate cancer cells using a luminescent cell nucleus stain composition.

13. The method according to claim 12, wherein the luminescent cell nucleus stain is Nuclear-Red stain.

14. A method of immobilising target prostate cancer cells from urine or a urine derived fluid on a substrate surface, the method comprising:

providing a sample of urine or a urine derived fluid;

providing a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome; and

contacting the sample of urine or a urine derived fluid with the one or more cell capture surface under conditions to bind at least some of the target prostate cancer cells or target prostate cancer cell- derived exosomes from the urine (if present) to the cell capture surface.

15. The method according to claim 14, wherein the prostate cancer cell or exosome selective binding agents comprise one or more PSMA antibodies or functional equivalents thereof.

16. The method according to any one of claims 1 to 15 wherein the cell capture surface comprises a functionalized fdm on the substrate and the one or more prostate cancer cell or exosome selective binding agent(s) is/are covalently bound to the functionalized film.

17. A device for selective capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid, the device comprising a substrate having one or more cell capture surface, each cell capture surface comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome.

18. A microfluidic device for selective capture of target prostate cancer cells or target prostate cancer cell-derived exosomes from urine or a urine derived fluid, the device comprising a substrate having one or more cell capture micro-channel, each cell capture micro-channel comprising one or more prostate cancer cell or exosome selective binding agent capable of binding one or more target prostate cancer cell or target prostate cancer cell-derived exosome.

19. The device according to claim 17 or the microfluidic device according to claim 18, wherein the cell capture surface comprises a functionalized fdm on the substrate and the one or more prostate cancer cell or exosome selective binding agent(s) is/are covalently bound to the functionalized fdm.

20. The device according to claim 17 or claim 19 or the microfluidic device according to claim 18 or claim 19, wherein the prostate cancer cell or exosome selective binding agents comprise one or more PSMA antibody or functional equivalent thereof.